This application is focused on the development of a simple and sensitive technology for the detection of CpG methylation in CpG islands of human cancer cells. CpG islands are short genomic segments in the promoter regions of most human genes that are enriched in the dinucleotide CpG. CpG islands are unmethylated in normal tissues but become progressively more methylated in cancer cells leading to repression of genes that control the cell cycle. Most current methods for the detection of CpG methylation depend on bisulfite treatment to allow discrimination between methylated and unmethylated cytosines, followed by PCR amplification of the DNA. Unmethylated cytosines are converted to uracils while methylated cytosines remain unchanged. Chemically converted DNA is then analyzed by a wide range of methods including sequencing or microarrays. Bisulfite treatment is technically difficult to perform, consistently causing extensive damage to sample DNA and inducing variability in these assays. The main goal of this application is to develop a methyl-CpG detection system that does not employ chemical modification of sample DNA by bisulfite treatment or PCR amplification. Signal generation will be based on Ribomed's proprietary Abscription. (Abortive Transcription) process in which DNA detectors called Abortive Promoter Cassettes (APCs) are bound to methyl-CpG sites via methyl-CpG specific probes. The proprietary Abscriptase enzyme will produce short RNA molecules from the APCs as signals for the presence of methylated CpGs. The Specific Aims will focus on methods to produce a detection reagent composed of a methyl-CpG binding protein covalently linked to an APC in high purity and at low cost. A system for capturing unmodified single-stranded CpG islands and reconstructing their methylation patterns will be validated in conjunction with the validation of the methyl-CpG binding probes. A method for interrogating a specific individual CpG site, and a method for measuring the overall level of methylation of a CpG island will be demonstrated. Successful completion of the proposed research will provide a simple and sensitive diagnostic method for assessing CpG methylation in CpG-islands with applications in cancer screening. The first proof of principle will focus on a segment of the p16 CpG island and CpG sites that have been intensively analyzed in the literature. Commercial products resulting from this research will be applied to the diagnosis of cancer, prognosis and monitoring of treatment. The detection methods developed in this project will exploit information about cancer related methylation signatures developed by other workers. Over the long term new detection kits will be marketed as more CpG island methylation signatures are validated by other workers. [unreadable] [unreadable] [unreadable]